1. Field of the Invention
The present invention relates to a method of comparing multiple samples of cell extracts for detecting differences in the protein composition of cells and cells extracts, and more particularly, to a method utilizing matched pair of labeling reagents for detecting such differences. By the expression samples of cell extracts is meant any sample that can be subject to the analysis method outlined in the present application.
2. Background of the Invention
Researchers studying various aspects of cell biology use a variety of tools to detect and monitor differences in cell structure, function and development. An essential part of studying cells is studying the differences and similarities in the protein composition between the different cell types, stages of development and condition. Determining differences in the protein content between normal and cancerous cells or wild type and mutant cells, for example, can be a valuable source of information and a valuable diagnostic tool.
Mixtures of proteins can be separated into individual components according to differences in mass by electrophoresing in a polyacrylamide gel under denaturing conditions. One dimensional and two dimensional gel electrophoresis have become standard tools for studying proteins. One dimensional SDS (sodium dodecyl sulfate) electrophoresis through a cylindrical or slab gel reveals only the major proteins present in a sample tested. Two dimensional polyacrylamide gel electrophoresis (2D PAGE), which separates proteins by isoelectric focusing, i.e., by charge, in one dimension and by size in the second dimension, is the more sensitive method of separation and will provide resolution of most of the proteins in a sample.
The proteins migrate in one- or two-dimensional gels as bands or spots, respectively. The separated proteins are visualized by a variety of methods; by staining with a protein specific dye, by protein mediated silver precipitation, autoradiographic detection of radioactively labeled protein, and by covalent attachment of fluorescent compounds. The latter method has been heretofore only able to be performed after the isoelectric focusing step of 2D PAGE. Immediately following the electrophoresis, the resulting gel patterns may be visualized by eye, photographically or by electronic image capture, for example, by using a cooled charge-coupled device (CCD) or a laser based image detector. To compare samples of proteins from different cells or different stages of cell development by conventional methods, each different sample is presently run on separate lanes of a one dimensional gel or separate two dimensional gels. Comparison is by visual examination or electronic imaging, for example, by computer-aided image analysis of digitized one or two dimensional gels.
However, each different sample in the separate gels must be prepared with exacting precision because no two gels are identical, the gels may differ one from the other in pH gradients or uniformity. The electrophoresis conditions from one run to the next may be different.
The drawbacks by running separate gels are partly overcome by a process disclosed in WO-96/33406, entitled “Difference gel electrophoresis using matched multiple dyes”, which is incorporated herein in its entirety. According to this known process the differences between multiple samples of proteins extracted for example, from different cells, are detected by labeling each sample of such proteins with a different one of a set of matched luminescent dyes. The matched dyes have generally the same ionic and pH characteristics but absorb and/or fluoresce light at different wavelengths, producing a different color fluorescence. In addition, the dyes should be similar in size. The thus labeled samples are then mixed together and co-electrophoresed on a single gel. The proteins common to each sample comigrate to the same position. Proteins that are different will migrate alone to different locations on the gel and will fluoresce different colors, thereby identifying which initial sample has one or more proteins which differ from the initial sample or samples.
The gel can be analyzed by a two (or more) wavelength fluorescence scanner, by a fluorescent microscope or by any known means for detecting fluorescence. An electronic detection system such as a laser scanning system with a photo multiplier tube or a charged-coupled device (CCD) camera and a white light source or light sources having predetermined wavelengths, two electronic images are made of the wet gel using different known filter sets to accommodate the different spectral characteristics of the labels. One image views fluorescence of the first dye using a first filter appropriate to filter out all light except that emitted at the wavelength of the first dye and the other image views fluorescence of the second dye using a second filter, appropriate to filter out all light except that emitted at the wavelength of the second dye. Exposure is about from a few milliseconds to 500 seconds. Each image can be considered as a grid-like array of pixel intensity values.
The thus obtained images are then analyzed according to WO-96/33406 by a commercially available software package that either will subtract the first image from the second to identify spots that are different, or, alternatively, the images may be divided to leave only the spots not common to both images. In subtracting the images, like spots will cancel each other, leaving only those that are different. In ratio analysis, like spots will provide a value of one. Differences will result in values greater than one or less than one.
The above described analysis step in the method known from the WO-96/33406 is sometimes very time-consuming.
The traditional method of 2D gel analysis applied on the electronic images obtained according to the technique disclosed in WO-96/33406 may also be used. According to this technique each electronic image is individually analyzed in order to detect spots. Corresponding spots from the two images are then matched, compared and further analyzed.
Several drawbacks can be identified using the traditional method when analyzing the electronic images.
One drawback is the risks that spots are mismatched, i.e. spots believed to be corresponding in the two images are not. A reason to that may be that the detection of the electronic image is performed by a sensitivity that is too low, i.e. exactly the same spots are not detected in both images.
Another drawback might be that the further analyses, e.g. calculation of the differential expression between the two spots, have some inaccuracy. This is due to that a parameter related to a spot is not calculated in exactly the same way in the two images, e.g. a volume that represents a spot is determined by integration of an analysis area having different boundaries in the two images.
The matching procedure, shortly described above, is also a tedious and interactive process introducing variation in results depending on who is performing the interactive steps.
The object of the present invention is to achieve a less time-consuming method of evaluating the electronic images obtained from the electrophoresed mixture.
Another object of the present invention is to achieve a higher degree of accuracy in the analysis of the electronic images where spots representing components of the cell extracts are detected.
One further object of the invention is to provide a method that is user friendly in that only few parameters need to be set in order to perform the measurements.
This in turn will result in that the variation of the result of the measurements between users will be eliminated.
Still another object of the invention is to achieve a higher degree of automation when performing the measurements according to the invention.
And still another object of the present invention is that the results obtained by using the method will be considered highly reliable.